Before 1985 at the Pitié-Salpêtrière Hospital in Paris (2,400 beds), resistance to cefotaxime in clinical isolates of Enterobacteriaceae involved only species producing inducible class 1 β-lactamase. Between November 1985 and April 1987, however, 62 isolates (57 of Klebsiella pneumoniae and five of Escherichia coli) showed decreased susceptibility to cefotaxime(mean MIC, 8–16 μg/mL). The transferability of cefotaxime resistance in E. coli K12 was demonstrated for 15 of 16 selected isolates. By isoelectric focusing using iodometric detection with 20 mg of ceftriaxone/100 mL and determination of substrate and inhibition profiles, three β-lactamases mediating cefotaxime resistance were identified as SHV-2(isoelectric point [pI] 7.6), CTX-1 (pI 6.3), and “SHV-2-type” or SHV-3 (pI 6.98). The three β-lactamases hydrolyzed penicillins and cephalosporins (including cefotaxime and ceftriaxone) and were therefore designated “extended broad-spectrum β-lactamases” (EBS-Bla). The enzymes conferred to derivatives a high level of resistance to amoxicillin, ticarcillin, piperacillin, and cephalothin and a decreased degree of susceptibility (i.e., MICs increased by 10-to 800-fold) to cefotaxime, ceftriaxone, ceftazidime, and aztreonam. These β-lactamases did not affect the activity of cephamycins (cefoxitin, cefotetan, moxalactam) or imipenern. Synergy between clavulanate or sulbactam (2 μg/mL) and amoxicillin was greater against derivatives producing EBS-Bla than against those producing TEM-1, TEM-2, or SHV-I; this synergy was greater with clavulanate than with sulbactam against derivatives producing SHV-2 and the SHV-2-type enzyme but was similar with clavulanate and sulbactam against those producing CTX-1. A double-disk synergy test performed with cefotaxime and Augmentin disks (placed 30 mm apart, center to center) seemed a useful and specific test for the detection of strains producing EBS-Bla.